KR20070115356A - Natural anti-virus using weissella sp and composition comprising thereof - Google Patents

Abstract

A natural antiviral agent by using Weissella sp. is provided to improve inhibitory effects on virus infection and safety of application by removing side effects, so that the antiviral agent is applicable to food, feed, drink, functional food and medicines. A natural antiviral agent contains Weissella sp. which is selected from Weissella koreensis(KCTC 10400BP), Weissella hanii, Weissella kimchii, Weissella soli and Weissella confusa and isolated from kimchi, its pulverized solution, cultured medium, concentrated solution, dried product or a mixture thereof. An antiviral composition comprises the Weissella koreensis(KCTC 10400BP), wherein the daily dosage of the antiviral composition is 0.0001-100 mg/kg.

Description

Natural anti-viral agent using Bisella genus strain and composition comprising same {Natural anti-virus using Weissella sp and Composition comprising

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a natural antiviral agent and a composition comprising the same, in particular, Kimchi lactic acid bacteria appearing during the ripening process of kimchi, more specifically, the virus is produced using a strain of Bisella genus, a predominant species among kimchi lactic acid bacteria, influenza The present invention relates to a natural antiviral agent having a high inhibitory effect against infection of viruses and avian viruses and having no side effects, and having high safety and compositions such as beverages, feedstuffs, foodstuffs, functional foods and pharmaceuticals containing the same.

[Private Technology]

The influenza virus that causes the flu every year is an RNA virus with an envelope of about 1 / 10,000 mm in diameter. It is classified into three types of A, B, and C from the difference of antigenicity, but it is type A, B which shows epidemic spread. On the particle surface of these viruses, two types of glycoproteins, erythrocyte aggregates (Hermagglutinin, HA) and neuramidase (NA), protrude onto spikes, and there are eight segmented gene RNAs inside. Hemagglutinin and neuraminidase on the surface of the virus cause mutations in the same subtype, with new antigenic variants appearing each year.

Influenza viruses released by cough droplets invade human noses and mouths and are adsorbed to the mucosal epithelial cells of the human respiratory tract by spike-like glycoprotein erythrocyte aggregates on the surface of the virus and invade and then proliferate. Influenza virus infection causes symptoms such as sudden fever, headache, joint pain, and general boredom for several days. Before and after this, respiratory symptoms such as cough, sore throat, runny nose and stuffy nose appear. It is different from the so-called cold, and its infectivity is strong, which is characterized by an explosive outbreak in a short time.

In addition, the hemagglutinin protein structure of the influenza virus is mutated every year, and the lack of antibodies caused by past infections is a factor that spreads the infection.

Avian Inflenza Virus, which is a recent problem among influenza viruses, infects various kinds of birds such as chickens, turkeys, ducks, and wild birds to represent Pathogenic Avian Influenza. Avian influenza is an acute viral infectious disease that spreads rapidly and causes many harms to poultry such as chickens and turkeys. It is a viral disease that causes the greatest damage and threat to the poultry industry worldwide, and its ramifications are not limited to the poultry industry. It has been mentioned as a disaster of all mankind.

In fact, in China and Southeast Asian countries, highly pathogenic avian influenza caused by influenza virus is continuously occurring, and if the current trend is taken, there is a possibility that another problem will be caused by new mutations rather than eradicating influenza in these countries. . In Indonesia, it is not possible to control the epidemic, and in China, many kinds of high-pathogenic influenza viruses are separated from ducks and wild birds, so the spread is faster than the rate of disease suppression, so there is no alternative except vaccination. I'm doing it. In Vietnam and Thailand, cases of continuous human infections and deaths are reported and there is no sign of calming down.

Avian influenza is classified into three types of high pathogenic, low pathogenic and non-pathogenic according to pathogenicity. Among these, highly pathogenic avian influenza (HPAI) is classified as one of the most dangerous livestock infectious diseases classified as list A level by the International Water Bureau, International Animal Health Organization. .

In addition, avian influenza is classified according to two types of glycoproteins, erythrocyte agglutinates (Hermagglutinin (HA)) and neuramidase (NA) on the particle surface of the virus, and a total of 135 kinds of avian influenza viruses are reported. It is becoming. The causative agent of highly pathogenic avian influenza is the type A virus, which has a hemagglutinin serotype and a neuraminidase serotype. Avian influenza, which has recently caused great damage in Europe and Asia, is a disease caused by the highly pathogenic H5N1 influenza virus. When chickens are infected by the influenza virus, more than 80% of them are reported to be dying due to respiratory distress.In contrast, most avian influenza viruses do not directly infect humans, whereas H5N1 influenza virus causes human diseases. It is reported.

Currently, there are three methods that can be used to control avian influenza in poultry farms. The first stop is biosecurity, the second stop is vaccination, and the third stop is the family's own immune system. The blockade is judged to be practically ineffective as the global expansion of avian influenza shows, and as the mortality caused by highly pathogenic avian influenza, such as H5N1 type, is shown, the family's own immune system has been found to be ineffective against highly pathogenic avian influenza. to be. Therefore, there is a need for the development of a vaccine that is a secondary stop wire, that is, a therapeutic agent for avian influenza, or a therapeutically improving agent of a complex function that dramatically boosts the immune system.

Avian influenza viruses, like other influenza viruses, bind to endosomes bound to sugar chain receptors present on the surface of target cells and are surrounded by endosomes and invade into cells by fusion of viral and endosomal membranes, followed by transcription and replication of viral genes. And by proliferation by forming a progeny virus particle by sprouting from the host cell membrane. In order to inhibit the infection of influenza virus, inhibition of adsorption to epithelial cells, inhibition of invasion into cells, inhibition of gene transcription and replication, inhibition of protein synthesis and release from cells may be considered. Has become a major target of antiviral agents.

However, avian influenza virus has so many different serotypes, and each serotype does not become cross-immune or weakly cross-immune, which prevents the influenza virus infections of different serotypes and is well mutated. There is a difficulty.

Until now, amantadine, rimantadine, zanamiville and the like have been developed as antiviral agents, but side effects such as hypersensitivity, mental nerve symptoms, digestive system symptoms, and autonomic nervous system symptoms have been reported. In addition, suppressing infection by vaccination is less effective if the type of the virus is inconsistent with the type of the virus. There is a limit to the prevention of vaccines, and effective treatments and precautions against influenza viruses are still inadequate. It is a state.

For this reason, it is desired to develop an anti-influenza virus agent which is highly effective in inhibiting infection, has no problem in stability, and which can be used on a daily basis.

An object of the present invention is to provide a natural antiviral agent having high safety against infections such as influenza virus and avian influenza virus and having no side effects.

In addition, an object of the present invention is to provide a composition that can be applied to a variety of products, such as beverages, feed, food, functional foods and pharmaceuticals containing the natural antiviral agent as an active ingredient.

In order to achieve the above object, the present invention is a kimchi lactic acid bacteria selected from the group consisting of bacteria of the genus Bisella, and cell disruption, and a culture solution containing the cells, a concentrate thereof, and dried products thereof, and mixtures thereof. It relates to any one antiviral agent.

Preferably, the strain of the genus Bisella is Weissella koreensi , Weissella hanii , Weissella kimchii , Weissella soli , Weissella soli , Weissella confusa ) and mixed lactic acid bacteria, characterized in that any one selected from the group consisting of, more preferably Weissella Koreensis (Weissella Koreensis) KCTC 10400BP. In addition, a preferred antiviral effect of the present invention is avian influenza virus. The bisella corriensis KCTC10400BP is a strain described in Patent 0454591, which was deposited on December 10, 2002 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 1040BP).

The antivirus according to the present invention can inhibit and treat infection of common viruses, more preferably avian influenza virus.

In addition, the present invention is a beverage, food and functional food containing the antiviral agent; feed; And pharmaceuticals.

Hereinafter, the present invention will be described in more detail.

The present invention is a kimchi lactic acid bacteria appearing during the ripening process of kimchi, preferably the bisella genus strains that are the predominant species of kimchi lactic acid bacteria, culture medium containing the cells, concentrated solution of the culture solution, dried products of the culture medium, cells, cell disruption and these It provides any one natural antiviral agent selected from the group consisting of.

In the natural antiviral agent according to the present invention as described above, the Weissella genus Kimchi lactic acid bacteria are Weissella koreensis ( Weissella koreensis ), Weissella hanii ( Weissella hanii ), Weissella kimchii ( Weissella kimchii ) , Weissella soli ( Weissella soli ), Weissella confusa ( Weissella confusa ) and is preferably any one selected from the group consisting of these mixed lactic acid bacteria, the Weissella genus Kimchi lactic acid bacteria are bisella corriensis ( the more preferred Weissella koreensis) and by N-Sys Cellar Corey (Weissella most preferred is koreensis KCTC 10400BP).

In the present invention, the pickled radish, cabbage and cucumber, or salted or pickled in the seasoning of pepper, garlic, green onions, ginger, salted fish, etc. and then aged by the production of lactic acid in the fermentation process of kimchi fermented at low temperature The bacteria of the emerging kimchi lactic acid bacteria, preferably of the genus Bisella, serve as excellent antiviral agents against influenza viruses and avian influenza viruses, and the antiviral spectrum can serve as a broad multifunctional natural antiviral agent. .

Conventional antiviral patents using lactic acid bacteria emphasize the efficacy of the culture medium, ie, bacteriocin, excluding the cells of lactic acid bacteria, but according to the present invention, the effect of inhibiting the virus infection by the cells itself is greater than the effect of the culture medium without cells, In particular, cell lysate was shown to be effective. This shows that it has antiviral effect even in the state of probiotics rather than live bacteria. It is useful for developing various formulations in the future, and it has high industrial efficiency in increasing production cost or production yield compared to using bacteriocin. have.

In addition, the present invention provides a beverage, feed, food, functional food and pharmaceutical products containing the natural antiviral agent.

In the present invention, the drink refers to a generic term for drinking to quench thirst or enjoy the taste, and includes alcoholic beverages, soft drinks, water, syrup, tea, coffee, fruit drinks, and the like, and include lactic acid bacteria drinks. The lactic acid bacteria drink refers to a beverage in which lactic acid bacteria are cultured and lactic acid fermentation is added by diluting with sterilized water and adding sugar and flavoring. The beverage is not particularly limited to other ingredients except for containing the probiotic as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. In the present invention, the feed refers to a substance for maintaining the life of the livestock and supplying organic or inorganic nutrients necessary for producing milk, meat, eggs, fur, and the like. The feed can be classified into various kinds according to nutritional value, main ingredient, distribution, moisture content and processing type, etc. The feed of the present invention includes feed, thick feed, supplementary feed, protein feed, starch feed, fat feed, and fiber feed. Include.

In the present invention, the food means a natural product or a processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process, for example, fruits, vegetables, and fruits. Dried or cut products of fruits, vegetables, fruit juices, vegetable juices, mixed juices or chips, noodles, livestock processed foods, fish processed foods, dairy foods, fermented foods, legumes foods, grain foods, microbial fermented foods, confectionery bakery , Seasonings, processed meats, acidic beverages, licorice, herbs, etc., but is not limited thereto. The food of the present invention is not particularly limited to other ingredients except for containing the natural antiviral agent as an essential ingredient in the indicated ratio, and may include various flavors or carbohydrates as additional ingredients, such as ordinary food, but is not limited thereto. It doesn't happen. The natural antiviral agent may be added to raw materials in the manufacture of foods, beverages, health drinks, gums, teas, vitamin complexes, and health functional foods, or by appropriate mixing with cooked foods. Can be added with food supplement additives. At this time, the amount of the natural antiviral agent in the food or beverage may be added at 0.01 to 90% by weight of the total food weight, in the case of a beverage in the ratio of 0.02 to 20 g, preferably 0.5 to 10 g based on 100 mL Can be added.

Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food processed and designed to fully express the gymnastics function related to the living body.

In the present invention, the medicine may be used in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. It may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament. In the context of the pharmaceutical dosage forms of the probiotic actives according to the invention, the forms of their pharmaceutically acceptable salts can be used, as well as alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection. Preferred dosages of the probiotic of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but will be appropriately selected by one of ordinary skill in the art. Can be. However, for the desired effect, the probiotic of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes, and all modes of administration can be expected. The medicine may also contain various supplemental herbal medicines, vitamins, minerals, dietary fiber and other medicinal and food supplements (eg, excipients, enhancers, coatings, etc.) as necessary.

Hereinafter, the embodiments will be described in detail. The following examples are merely illustrative of the present invention, and thus the scope of the technical spirit of the present invention is not changed or reduced.

EXAMPLE

Example 1 Preparation of Antiviral Agents

1-1: Cultivation of Bicellar corriensis

In order to culture the kimchi lactic acid bacteria, Weissella Koreensis KCTC 10400BP was used among the kimchi lactic acid bacteria.

After 1 L of MRS broth (Difco) was autoclaved at 121 ° C. for 15 minutes, the cells were inoculated with Weissella Koreensis KCTC 10400BP and shaken at 30 ° C. for 24 hours.

1-2: Manufacture of bicella coensis cell lysate

A portion of the culture was centrifuged at 5,000 X g for 5 minutes to discard the supernatant and to take up the cells. The harvested cells were diluted in 50 ml of PBS buffer (Phosphate buffered saline, pH 7.4), and the cells were disrupted by an ultrasonic crusher and the destruction of cells was observed under a microscope.

1-3: Cell-free culture preparation

The supernatant centrifuged above was passed through a 10,000 Da membrane, and the filtrate was collected and concentrated again to 1,000 Da membrane, and again obtained bacteria-free state with a pore size 0.22 ㎛ membrane.

Example 2: Isolation of Avian Flu Virus

Fecals from suspicion of virulent pathogenic avian influenza were collected from a laying hen farm near Gwangju, Gyeonggi-do. The collected feces were immediately transported to the laboratory, suspended in sterile PBS buffer (pH 7.4), filtered with 0.45 μm filter paper, and filtered again with 0.22 μm filter paper to prevent bacterial infection. The filtrate was serially diluted with sterile PBS buffer and then infected with monolayer cultured Madin-Darby Canine Kidney (MDCK, KCLB 10034) cells. When cultured at 5% CO ₂ , 37 ℃, 70 ~ 80% of cytopathic effect (Cytophatic effect, CPE) is observed, harvested and centrifuged (4 ℃, 10,000rpm, 15 min) to obtain supernatant and cell sediment. The supernatant was stored at -70 ° C.

Identification of influenza virus was confirmed to be influenza virus using Real-Time Reverse Transcripase PCR after RNA was separated from the supernatant stored above using Viral RNA kit (Qiagen Inc.). PCR primer sequences used therein are shown in Tables 1 and 2 below, and are shown in SEQ ID NOs: 1-6. FAM and TAMRA linked to the 5 'end and the 3' end of the sequences shown in SEQ ID NOs: 3 and 6 in Table 1 and Table 2 are fluorescent dyes exhibiting fluorescence. The PCR is a method that can directly quantify the PCR product amplified by the probe in real time by performing the PCR with two primers using a real time PCR machine.

TABLE 1

PCR primers for influenza A virus

designation Sequence (5'-3 ') SEQ ID NO Primer M + 25 AGA TGA GTC TTC TAA CCG AGG TGG One Primer M-124 TGC AAA AAC ATC TTC AAG TCT CTG 2 probe M + 64 FAM-TCA GGC CCC CTC AAA GCC GA-TAMRA 3

TABLE 2

PCR primers for Avian H5 virus

designation Sequence (5'-3 ') SEQ ID NO PrimerH5 + 1456 ACG TAT GAC TAT CCA CAA TAC TCA G 4 PrimerH5-1685 AGA CCA GCT ACC ATG ATT GC 5 probe H5 + 1637 FAM-TCA ACA GTG GCG AGT TCC CTA GCA-TAMRA 6

Example 3 Anti-Avian Flu Effect of Bisella Corriensis

Madin-Darby Canine Kidney (MDCK, KCLB 10034) cells highly susceptible to influenza virus were distributed from Korea Cell Line Bank. Cell cultures were incubated at 5% CO ₂ , 37 ° C using minimal nutrient medium (Minimum Essential Medium; GIBCO BRL, USA) containing 10% fetal bovine serum (FBS).

플레이트에 단층으로 80%이상 자란 MDCK 세포주에 PBS(pH 7.4)로 10^-3 비율로 희석된 조류 독감 바이러스와 동일한 완충용액으로 희석된 바이셀라 코리엔시스 배양액, 바이셀라 코리엔시스 균체 및 바이셀라 코리엔시스 균체파쇄액을 혼합하여 감염시킨 후 3일간 배양하고 0.6% agarose로 고정화하고 neutral red로 플라크를 염색하여 형성된 플라크를 계수하였다. 그 결과를 표 3에 제시하였다. 억제율은 다음식을 통해 계산하였다. 모든 실험은 triplicate로 실험하였고 평균을 계산하였다.

Bisella corriensis culture, bisella corriensis cells, and bisella corriensis diluted with the same buffer as avian influenza virus diluted ^10-3 in PBS (pH 7.4) in MDCK cell lines grown more than 80% in monolayers on plates. After mixing and infecting the cell lysate solution, the cells were cultured for 3 days, immobilized with 0.6% agarose and stained with neutral red to count the plaques formed. The results are shown in Table 3. Inhibition rate was calculated by the following equation. All experiments were triplicated and averaged.

TABLE 3

Plaques % Inhibition Control (virus control) 145 0.0 Culture medium that does not contain cells 113 22.1 Culture medium containing cells 47 67.6 Cell lysate 24 83.5 Cells themselves 38 73.8

As shown in Table 3, 145 plaques were formed in the control group that did not include bisella corriensis, while very strong against avian influenza virus in the presence of culture, cells, or cell disruption containing bisella corriensis. It was found that there is antiviral activity.

According to the results, it can be seen that the bacteria and the like of kimchi lactic acid bacteria according to the present invention has a very excellent antiviral effect. And the experiment was omitted for the stability of the kimchi lactobacillus culture medium according to the present invention, kimchi is ingested in humans for a long time and its safety has been proved that the stability of the kimchi lactobacillus culture medium in the present invention without a separate experiment can be said that have.

As described above, the culture medium containing the bacteria of Weissella Koreensis , the dominant species among the kimchi lactic acid bacteria according to the present invention, the concentrated solution of the culture solution, the dried product of the culture solution, cells, cell disruption solution and mixtures thereof are very excellent It has viral activity, and if used, it can safely and effectively impart antiviral effects to beverages, feed, food, medicines, and cosmetics.

<110> OVOBIO INC. <120> Natural anti-virus using Weissella sp and Composition comprising          about <130> DPP-2006-1378-KR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Influenza A virus M + 25 <400> 1 agatgagtct tctaaccgag gtgg 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> Influenza A virus M-124 <400> 2 tgcaaaaaca tcttcaagtc tctg 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Influenza A virus M + 64 <400> 3 tcaggccccc tcaaagccga 20 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Avian H5 + 1456 <400> 4 acgtatgact atccacaata ctcag 25 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Avian H5-1685 <400> 5 agaccagcta ccatgattgc 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Avian H5 + 1637 <400> 6 tcaacagtgg cgagttccct agca 24

Claims (6)

As kimchi lactic acid bacteria, cells of the genus Bisella, and cell disruption liquid Any one antiviral agent selected from the group consisting of a culture solution containing the cells, a concentrate thereof, and a dried product thereof, and a mixture thereof. According to claim 1, Weissella koreensis , Weissella hanii , Weissella kimchi ( Weissella) kimchii ), Weissella soli , Weissella sofusa confusa ) and mixed lactic acid bacteria thereof, any one selected from the group consisting of antiviral agents. The method of claim 2, wherein the strain is Weissella Weissella Koreensis ) Antiviral agent for avian influenza KCTC 10400BP. Beverages, foods and functional foods comprising the antiviral agent according to any one of claims 1 to 3. A feed comprising the antiviral agent according to any one of claims 1 to 3. A pharmaceutical product comprising the antiviral agent according to any one of claims 1 to 3.